U.S. patent application number 11/365135 was filed with the patent office on 2006-06-29 for enzymatic methods for modulating the levels of organic sulfur compounds in plants.
This patent application is currently assigned to Pioneer Hi-Bred International, Inc.. Invention is credited to Changjiang Li, Bo Shen, Mitchell C. Tarczynski.
Application Number | 20060143733 11/365135 |
Document ID | / |
Family ID | 36216021 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060143733 |
Kind Code |
A1 |
Tarczynski; Mitchell C. ; et
al. |
June 29, 2006 |
Enzymatic methods for modulating the levels of organic sulfur
compounds in plants
Abstract
Methods for modulating levels of at least one organic sulfur
compound in plants are provided. Also provided are plants and seeds
produced by the methods. The methods comprise stably transforming a
plant with a DNA construct encoding a cystathionine gamma synthase
enzyme, a serine acetyl transferase enzyme and/or other sulfur
assimilating enzymes capable of altering the level of at least one
organic sulfur compound.
Inventors: |
Tarczynski; Mitchell C.;
(West Des Moines, IA) ; Li; Changjiang; (Johnston,
IA) ; Shen; Bo; (Johnston, IA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL, INC.
7250 N.W. 62ND AVENUE
P.O. BOX 552
JOHNSTON
IA
50131-0552
US
|
Assignee: |
Pioneer Hi-Bred International,
Inc.
|
Family ID: |
36216021 |
Appl. No.: |
11/365135 |
Filed: |
March 1, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10108739 |
Mar 29, 2002 |
7038109 |
|
|
11365135 |
Mar 1, 2006 |
|
|
|
60367333 |
Mar 29, 2001 |
|
|
|
Current U.S.
Class: |
800/278 ;
800/320.1 |
Current CPC
Class: |
C12N 15/8243 20130101;
C12N 15/8242 20130101 |
Class at
Publication: |
800/278 ;
800/320.1 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01H 1/00 20060101 A01H001/00 |
Claims
1. A method for modulating biosynthesis of at least one organic
sulfur compound in the seed of a monocotyledonous plant, the method
comprising stably transforming the plant with a DNA construct
comprising a nucleic acid encoding a plant cystathionine gamma
synthase nucleic acid, wherein the nucleic acid is operably linked
to a non-seed preferred promoter that drives expression in the
plant and wherein the level of the at least one organic sulfur
compound is altered.
2. The method of claim 1, wherein the promoter is a constitutive
promoter.
3. The method of claim 1, wherein the promoter is a
tissue-preferred promoter for a tissue other than seed.
4. The method of claim 1, further comprising transforming the plant
with a second DNA construct comprising a second nucleic acid
encoding a plant serine acetyl transferase nucleic acid, wherein
the second nucleic acid is operably linked to a non-seed preferred
promoter that drives expression in the plant.
5. The method of claim 1, wherein the organic sulfur compound is
cysteine or methionine.
6. A monoctyledonous plant with seed having increased levels of at
least one organic sulfur compound, the plant having stably
transformed into its genome a DNA construct comprising a nucleic
acid encoding a plant cystathionine gamma synthase nucleic acid,
wherein the nucleic acid is operably linked to a non-seed preferred
promoter that drives expression in the plant.
7. The plant of claim 6, wherein the promoter is a constitutive
promoter.
8. The plant of claim 6, wherein the promoter is a tissue-specific
promoter for a tissue other than seed.
9. The plant of claim 6, further comprising a second nucleic acid
encoding a plant serine acetyl transferase nucleic acid, wherein
the second nucleic acid is operably linked to a non-seed preferred
promoter that drives expression in the plant.
10. The plant of claim 6, wherein the organic sulfur compound is
cysteine or methionine.
11. The plant of claim 6, wherein the plant is maize.
12. Seed of the plant of claim 11.
13. A method for modulating biosynthesis of at least one organic
sulfur compound in the seed of a monocotyledonous plant, the method
comprising stably transforming the plant with a DNA construct
comprising a nucleic acid encoding a plant serine acetyl
transferase nucleic acid, wherein the nucleic acid is operably
linked to a non-seed preferred promoter that drives expression in
the plant and wherein the level of the at least one organic sulfur
compound is altered.
14. The method of claim 13, wherein the promoter is a constitutive
promoter.
15. The method of claim 13, wherein the promoter is a
tissue-specific promoter for a tissue other than seed.
16. A monoctyledonous plant with seed having increased levels of at
least one organic sulfur compound, the plant having stably
transformed into its genome a DNA construct comprising a nucleic
acid encoding a plant serine acetyl transferase nucleic acid,
wherein the nucleic acid is operably linked to a non-seed preferred
promoter that drives expression in the plant.
17. The plant of claim 16, wherein the promoter is a constitutive
promoter.
18. The plant of claim 16, wherein the promoter is a
tissue-preferred promoter for a tissue other than seed.
19. The plant of claim 16, wherein the organic sulfur compound is
cysteine or methionine.
20. The plant of claim 16, wherein the plant is maize.
21. Seed of the plant of claim 20.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/108,739, filed Mar. 29, 2002, and which
claims the benefit of U.S. Provisional Application No. 60/367,333,
Mar. 29, 2001, all of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to the genetic manipulation of plants,
particularly to enzymatic methods for altering sulfur metabolism in
plants and plant seeds.
BACKGROUND OF THE INVENTION
[0003] Sulfur in its reduced form plays an important role in plant
metabolism, being involved in the biosynthesis of a wide range of
primary and secondary sulfur-containing metabolites. In plants,
sulfur metabolism includes the uptake of sulfate from the
environment, assimilation into organic compounds, and channeling
into proteins and secondary substances.
[0004] Plants and microorganisms are able to reduce sulfate to
sulfide for synthesis of the thiol group of cysteine. The first
step is the activation of sulfate by ATP sulfurylase, forming
5'-adenylylsulfate (APS). APS reductase acts upon APS to generate
sulfite, and sulfite reductase converts this sulfite to hydrogen
sulfide. This hydrogen sulfide provides the thiol group to
cysteine, while the carbon portion of cysteine comes from the
serine branch of the pathway. In this branch, serine is converted
to O-acetylserine by serine acetyltransferase. Cysteine synthase
then catalyzes the reaction of O-acetylserine and hydrogen sulfide
to form cysteine.
[0005] Cystathionine gamma synthase catalyzes the reaction between
O-phosphohomoserine and cysteine, wherein the cysteine donates a
thiol group to O-phosphohomoserine, thereby forming
cystathionine.
[0006] Methionine and sulfur-containing vitamins such as biotin or
thiamine are essential in human nutrition. Sulfur-mediated
functions include electron transport in Fe/S-clusters, structural
and regulatory roles via protein disulfide bridges, and catalytic
centers. Additionally, secondary sulfur compounds include signaling
molecules, anti-carcinogens and atmospheric compounds. See Hell
(1997) Planta 202:138.
[0007] Often plant protein is deficient in the sulfur amino acids,
especially methionine, as well as other essential amino acids such
as lysine and tryptophan. As a result, diets must be supplemented
with these amino acids in order to provide a balanced diet. A goal
of plant breeding has been to increase the amount of sulfur amino
acids present in the seed.
[0008] A number of methods have been described for increasing
sulfur amino acid content of plants. Some of these methods provide
for the overexpression of a high methionine seed storage protein,
which entails overexpressing the seed storage protein in a
transformed plant. Other methods have attempted seed specific
expression of synthetic enzymes in the methionine pathway. Still
other methods have focused on enzymatic modification of amino acids
and capturing these amino acids in transgenic seed storage
proteins. However, these methods have met with limited success.
There is therefore a need for an effective and direct method of
producing significant levels of the sulfur amino acids in plants
and plant seeds.
SUMMARY OF THE INVENTION
[0009] An object of the present invention is to provide methods for
increasing the nutritional value of plants.
[0010] Another object of the present invention is to provide plants
and plant parts having increased nutritional value.
[0011] Another object of the present invention is to provide plants
and plant parts having increased levels of organic sulfur
compounds.
[0012] Another object of the present invention is to provide plants
and plant parts having increased levels of methionine.
[0013] Another object of the present invention is to provide plants
and plant parts having increased levels of cysteine.
[0014] In accordance with the present invention, methods for
modulating the level of at least one organic sulfur compound in
plants are provided. Also provided are plants, plant tissues, plant
seeds and plant cells produced by the methods. The methods comprise
stably transforming a plant with a DNA construct encoding a
cystathionine gamma synthase and/or serine acetyl transferase
enzyme.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 sets forth the biosynthesis for the organic sulfur
compounds cysteine and methionine with the proposed modifications
of the invention.
[0016] FIG. 2 sets forth the sulfur amino acid level, on a percent
dry weight basis, of certain cystathionine gamma synthase
transformed lines.
[0017] FIG. 3 sets forth the sulfur amino acid level, on a mol
percent level, of certain cystathionine gamma synthase transformed
lines.
[0018] FIG. 4 sets forth the sulfur amino acid level, on a percent
dry weight basis, of certain serine acetyl transferase transformed
lines.
[0019] FIG. 5 sets for the sulfur amino acid level, on a mol
percent level, of certain serine acetyl transferase transformed
lines.
[0020] FIG. 6 sets for the sulfur amino acid level, on a percent
dry weight basis, of the segregating seed (T2) of certain serine
acetyl transferase transformed lines.
[0021] FIG. 7 sets for the sulfur amino acid level, on a mol
percent level dry weight basis, of the segregating seed (T2) of
certain serine acetyl transferase transformed lines.
DETAILED DESCRIPTION OF THE INVENTION
[0022] In accordance with the subject invention, compositions and
methods for modulating the biosynthesis of organic sulfur compounds
in plants, particularly sulfur amino acids, more particularly
cysteine and methionine, are provided. The methods involve
transforming a monoctyledonous plant with one or more nucleic
acid(s) encoding cystathionine gamma synthase or serine acetyl
transferase. The plant may also comprise one or more additional
nucleic acid(s) selected from nucleic acids encoding enzymes
involved in amino acid biosynthesis and sulfate reduction.
[0023] Previous attempts to modulate the biosynthesis of organic
sulfur compounds through enzymatic modification has focused upon
the enzymatic modification occurring in the plant seed. This is
because the plant seed is the desired source for the modulated
organic sulfur compound. Surprisingly, Applicants have discovered
that modulated expression of cystathionine gamma synthase and/or
serine acetyltransferase need not occur in a monocot plant seed in
order to modulate the biosynthesis of organic sulfur compounds in
the plant seed. Through non-seed specific enzymatic modulation of
cystathionine gamma synthase or serine acetyl transferase,
Applicants have increased the levels of methionine in a monocot
seed beyond those achieved through seed specific enzyme
modification. Perhaps most surprisingly, such increase in the seed
was accomplished without transgenic modification of the plant to
increase the expression of a seed storage protein containing the
desired amino acids.
[0024] By "organic sulfur compounds" is intended a compounds such
as cysteine, cystathionine, methionine, glutathione,
dimethylsulfoniopropionate (DMSP), SMM (Vitamin U), biotin, SAM,
Thiamine pyrophosphate (Vitamin B-1), Coenzyme A and sulfur
containing phytoalexins.
[0025] Sulfate reduction occurs in both roots and shoots of plants.
Most of the sulfur transported in the xylem to the leaves is in
non-reduced SO.sub.4.sup.2-. Some transport back to roots and other
parts of the plant occurs through phloem, and both free
SO.sub.4.sup.2- and organic sulfur compounds are transported. In
leaves, the process of sulfate reduction occurs in chloroplasts. In
roots, most or all of the process occurs in proplastids.
[0026] Therefore, a preferred embodiment of the present invention
is to provide cystathionine gamma synthase and/or serine acetyl
transferase expression in the non-seed tissues of the plant where
sulfate reduction is most active. This can be accomplished through
tissue specific expression of these enzymes in non-seed tissue,
such as leaves, roots or shoots or by constitutive expression of
these enzymes. Constitutive expression will result in expression in
both seed and non-seed tissue. Other methods can be used for
increasing the activity of the cystathionine gamma synthase and/or
serine acetyl transferase, such as protein engineering or DNA
shuffling.
[0027] Other nucleic acids encoding enzymes involved in sulfate
reduction or organic sulfur compound biosynthesis, such as cysteine
and methionine biosynthesis, can be utilized to shunt the pathway
in particular directions. For example, APS kinase can be
down-regulated to increase APS concentration for APS reductase. In
the same manner, an increase in methionine can be utilized as a
sulfur source for downstream sulfur containing compounds. For
example, glutathione is produced by the two step reaction of
glutamate+cysteine+ATP.fwdarw.gamma-glutamylcysteine+ADP+Pi,
followed by
gamma-glutamylcysteine+glycine+ATP.fwdarw.glutathione+ADP+Pi, which
reaction is catalyzed by glutathione synthetase. Another example is
the series of reactions converting methionine to
dimethylsulfoniopropionate (DMSP); the reactions converting
methionine to vitamins and co-vitamins such as biotin, SMM (Vitamin
U), SAM, Thiamine pyrophosphate (Vitamin B-1) and Coenzyme A; and
the series of reactions converting methionine to sulfur containing
phytoalexins that serve a pathogen defense function.
[0028] Also, antisense constructs for cystathionine gamma synthase
and/or serine acetyl transferase can be utilized to direct
biosynthesis into a particular product or to stop biosynthesis for
the build-up of a particular compound. For example, an antisense
construct for cystathionine gamma synthase can be used to shunt
reduced sulfur away from methionine production.
[0029] Any means for producing a plant comprising the cystathionine
gamma synthase or serine acetyl transferase are encompassed by the
present invention. Additional nucleic acid(s) of interest can also
be used to transform a plant at the same time as the cystathionine
gamma synthase and/or serine acetyl transferase (cotransformation).
The additional nucleic acid(s) of interest may code for other
enzymes in the methionine pathway that will further modify cysteine
or methionine levels or downstream sulfur containing compounds such
as sulfur containing vitamins. The additional nucleic acid can also
be introduced into a plant that has already been transformed with
the cystathionine gamma synthase and/or serine acetyl transferase
nucleic acid. Alternatively, transformed plants, one expressing the
cystathionine gamma synthase and/or serine acetyl transferase and
one expressing the additional nucleic acid, can be crossed to bring
the nucleic acids together in the same plant. Subsequent crosses or
transformations can bring additional sequences together in the
plant.
[0030] Enzymes involved in cysteine and methionine biosynthesis are
known in the art. See, for example, aspartokinase (Masakazu et al.
(1992) "Mutant Aspartokinase Gene," Japan Patent 1994062866-A 1
Mar. 8, 1994, Accession No. E06825; Omori et al. (1993) J.
Bacteriol. 175(3):785-794; Accession No. X60821; Moriya et al.
(1995) Japan Patent 1997070291-A 13 Mar. 18, 1997; Accession No.
E12770); aspartate semialdehyde dehydrogenase (Calzada, F. R. A.,
Direct Submission, Centro Nacional de Investigaciones Cientificas,
Avenida 25 esq. 158 reparto Cubanacan, Playa Ciudad de la Habana,
Codigo Postal 6990, CUBA (1997), Accession No. Y15281; Daniel et
al. (1993) J. Mol. Biol. 232 (2):468-483; Accession No. Z22554;
Chen et al. (1993) J. Biol. Chem.; Accession No. Z22554; Accession
No. U90239; Brakhage et al. (1990) Biochimie 72(10):725-734;
Accession No. Z75208; Gothel et al. (1997) Eur. J. Biochem. 244
(1):59-65; Accession No. Z75208); homoserine kinase (See number two
under aspartokinase, Accession No. X60821; Nakabachi et al. (1997)
Insect Biochem. Mol. Biol. 27:1057-1062; Accession No. AB004856;
Ryoichi et al. (1986) Japan Patent 1987232392-A 1 Oct. 12, 1987
(JP1986076298); Accession No. E01358; Sadao et al., Japan Patent
1993207886-A 4 Aug. 20, 1993; Accession No. D14072); threonine
synthase (see number two under aspartokinase, Accession No. X6082;
Accession No. Z46263; Rognes, S. E., Direct Submission, Oct. 24,
1994, to University of Oslo, Department of Biology, Blindern, 0316
Norway, Accession No. Z46263; Accession No. L41666; Clepet et al.
(1992) Mol. Microbiol. 6(21):3109-3119; Accession No. X65033
S50569; Cami, B., Direct Submission, Mar. 11, 1992, Laboratoire de
Chimie Bacterienne, Centre Nationale de la Recherche, Scientifique,
31 Chemin I. Aiguier, BP 71 13277 Marseille Cedex, FRANCE,
Accession No. X65033 S50569); cystathionine gamma synthase
(cystathionine gamma synthase) (Kim and Leustek (1996) "Cloning and
analysis of the gene for cystathionine gamma-synthase from
Arabidopsis thaliana," Plant Mol. Biol. 32 (6), 1117-1124, USA,
Accession No. AF069317; Locke et al., Direct Submission, Jun. 3,
1997, AG Biotechnology, DuPont AF Products, PO Box 80402,
Wilmington, Del. 19880-0402 USA, maize cystathionine gamma
synthase, Accession No. AF007785 and AF007786; rice cystathionine
gamma synthase (AF076495); potato cystathionine gamma synthase
(AF082891, AF082892, AF144102); soybean cystathionine gamma
synthase (AF141602); arabidopsis cystathionine gamma synthase
(U43709, U83500, X94756, AC027035, AC051630); cystathionine beta
Ivase (Bork et al. (1997) Plant Physiol. 115:864-864; Accession No.
AJ001148; Sienko, M., Direct Submission, Jun. 5, 1995, Marzena
Sienko, Genetics, Institute of Biochemistry and Biophysics,
Pawinskiego 5a, Warsaw O.sub.2-106, POLAND, Accession No. U28383;
Ravanel et al. (1995) Plant Mol. Biol. 29 (4):875-882; Accession
No. L40511); methionine synthase (Kurvari et al. (1995) Plant Mol.
Biol. 29:1235-1252; Accession No. U36197; Ravanel et al. (1998)
Proc. Natl. Acad. Sci. USA 95(13):7805-7812; Accession No. U97200;
Michalowski et al., Direct Submission, Jan. 12, 1997, Biochemistry,
University of Arizona, BioSciences West 513, Tucson, Ariz. 85721
USA, Accession No. U84889; Eichel et al. (1995) Eur. J. Biochem.
230 (3):1053-1058; Accession No. X83499); ATP sulfurylase (Murillo
et al. (1995) Arch. Biochem. Biophys. 323(1):195-204; Accession No.
U06275; Leustek et al. (1994) Plant Physiol. 105:897-902; Accession
No. U05218; Bolchi et al., Direct Submission, Jul. 28, 1997,
Scienze Biochimiche, Viale delle Scienze, Parma, PR 43100 ITALY,
Accession No. AF016305; Laue et al. (1994) J. Bacteriol.
176:3723-3729; Accession No. L26897; Laeremans et al. Accession No.
AJ001223); U.S. patent application Ser. No. 09/346,408 by DuPont
entitled, Sulfate Assimilation Proteins; soybean ATP sulfurylase
(BG156200, BG045264, BG041863, BF009916, BF009045, BE807651,
BE804451, BE556220, BE475509, BE473960, BE210444, BE191366,
AW458276, AW309791, AW278474, AW277946, AW277743, AW234410), tomato
ATP sulfurylase (BF113119, BF096727), rice ATP sulfurylase
(AB015204), brassica juncea ATP sulfurylase (AJ223498), brassica
oleracea ATP sulfurylase (AF195511), allium ATP sulfurylase
(AF212154), arabidopsis ATP sulfurylase (BE844959, BE039475,
AJ012586, U59737, AF198964, AF110407, U59738, AL161539, AP001300,
S68202); APS kinase (apk) (Korch et al. (1991) Mol. Gen. Genet.
229(1):96-108; Accession No. S55315; Arz et al. (1994) Biochim.
Biophys. Acta 1218 (3):447-452; Accession No. AF044285; Schiffmann
et al. "Isolation of cDNA clones encoding
adenosine-5'-phophosulfate-kinase (EC2.7.1.25) from Catharanthus
roseus (Accession No. AF044285) and an isoform (akn2) from
Arabidopsis (Accession No. AF043351)(PGR98-116)," Plant Physiol.
117 (3):1125 (1998); Accession No. AF044285; Jain et al. (1994)
Plant Physiol. 105:771-772; Accession No. U05238; Lee et al. (1998)
Biochem. Biophys. Res. Commun. 247:171-175; Accession No. U05238);
APS reductase (Speich et al. (1994) Microbiology 140 (Pt6):
1273-1284; Accession No. Z69372; Setya et al. (1996) Proc. Natl.
Acad. Sci. USA 93(23):13383-1338; Accession No. U56921; Bick et al.
(1998) Proc. Natl. Acad. Sci. USA 95(14):8404-8409; DuPont patent
application, Genes Encoding Sulfate Assimilation Proteins, PCT
publication number WO00/04161); PAPS reductase (Krone et al. (1991)
Mol. Gen. Genet. 225(2):314-319; Accession No. Y07525; Krone et al.
(1990) FEBS Lett. 260 (1):6-9; Accession No. Y07525;
Gutierrez-Marcos et al. (1996) Proc. Natl. Acad. Sci. USA
93:13377-13382; Accession No. U53865; Schwenn, J. D., Direct
Submission, Jul. 2, 1993, Ruhr-University-Bochum, Fac. Biology,
Biochemistry of Plants, Universitaetsstr. 150, D44780 Bochum,
GERMANY, Accession No. Z23169; see number five under ATP
sulfurylase, Accession No. AJ001223; Bussey et al. (1997) Nature
387 (6632 Suppl.):103-105; Accession No. U25840 U00094); sulfite
reductase (Accession No. Y07525; Accession No. Z23169; Hipp et al.
(1997) Microbiology 143 (Pt 9):2891-2902; Accession No. U84760;
Pott et al. (1998) Microbiology 144 (Pt 7):1881-1894; Accession No.
U84760; Bork et al. (1998) Gene 212 (1):147-153; Accession No.
Y10157; Mbeguie-A-Mbeguie et al. Accession No. AF071890; Bruehl et
al. (1996) Biochim. Biophys. Acta 1295:119-124; Accession No.
Z49217; Hummerjohann et al. (1998) Microbiology 144 (Pt
5):1375-1386; Accession No. AF026066; serine acetyltransferase
(Accession No. X80938; Accession No. D88529 and D88530; Saito et
al. (1995) J. Biol. Chem. 270 (27):16321-16326; Accession No.
D49535; DuPont patent application, Genes Encoding Sulfate
Assimilation Proteins, PCT publication number WO 00/04167; see also
arabidopsis serine acetyl transferase (U22964, L78443, X8288,
L78444, AF112303, U30298, L42212, U22964, L78443, X82888, Z34888
and X80938), soybean serine acetyl transferase (BF041806, BE802695,
AW234818, AI965408, AI495784, AI437954), tomato serine acetyl
transferase (BF176520, BF098353), allium serine acetyl transferase
(AF212156, AB040502) and cotton serine acetyl transferase
(A1725434); cysteine synthase (Hesse et al. (1998) "Isolation of
cDNAs encoding cytosolic (Accession No. AF044172) and plastidic
(Accession No. AF044173) cysteine synthase isoforms from Solanum
tuberosum (PGR98-057)," Plant Physiol. 116:1604, Accession No.
AF044173; Brander et al. (1995) Plant Physiol. 108:1748-1748;
Accession No. X85803; Topczewski et al. (1997) Curr. Genet. 31
(4):348-356; Accession No. U19395); gamma glutamylcysteine synthase
(Powles et al. (1996) Microbiology 142 (Pt 9):2543-2548; Accession
No. U81808 L75931; Accession No. AL031018; EU Arabidopsis
sequencing project, Direct Submission, Jul. 3, 1998, at the
Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18a, D-82152
Martinsried, FRG, Accession No. AL031018); glutathione synthetase
(Okumura et al. (1997) Microbiology 143 (Pt 9):2883-2890; Accession
No. D88540; Inoue et al. (1998) Biochim. Biophys. Acta 1395
(3):315-320; Accession No. Y13804; Accession No. Y10984; Accession
No. U22359).
[0031] Variants and functional fragments, including shufflents, of
the above enzymes, specifically including cystathionine gamma
synthase and serine acetyl transferase, may be utilized. It is only
required that the enzymes have an activity sufficient to modulate
the level of a particular organic sulfur compound in a plant.
Variants can be produced by methods known in the art. Variant
proteins include those proteins derived from the native protein by
deletion (so-called truncation), addition, or substitution of one
or more amino acids at one or more sites in the native protein.
Additional sequences for use in the invention may be obtained by
screening DNA libraries or sequence databases of plants or other
species. Some of the RNA partial sequences and precursors listed
above may be used to screen DNA libraries to obtain full length
genomic or cDNA sequences.
[0032] Amino acid sequence variants of the polypeptide can be
prepared by mutations in the cloned DNA sequence encoding the
native protein of interest. Methods for mutagenesis and nucleotide
sequence alterations are well known in the art. See, for example,
Walker and Gaastra, eds. (1983) Techniques in Molecular Biology
(MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol.
154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview,
N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein;
herein incorporated by reference. Guidance as to appropriate amino
acid substitutions that do not affect biological activity of the
protein of interest may be found in the model of Dayhoff et al.
(1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res.
Found., Washington, D.C.), herein incorporated by reference.
Conservative substitutions, such as exchanging one amino acid with
another having similar properties, may be preferred.
[0033] The cystathionine gamma synthase and serine acetyl
transferase nucleic acids, as well as any additional genes of
interest, can be optimized for enhanced expression in plants of
interest. See, for example, EPA0359472; WO91/16432; Perlak et al.
(1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al.
(1989) Nucleic Acids Res. 17:477-498. In this manner, the nucleic
acids can be synthesized utilizing plant-preferred codons. See, for
example, Murray et al. (1989) Nucleic Acids Res. 17:477-498, the
disclosure of which is incorporated herein by reference. In this
manner, synthetic nucleic acids can also be made based on the
distribution of codons a particular host uses for a particular
amino acid.
[0034] Another method for obtaining modified enzymes that can alter
the level of at least one organic sulfur compound is by sequence
shuffling. Sequence shuffling is described in PCT publication No.
96/19256. See also, Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509. Libraries of recombinant polynucleotides are
generated from a population of related sequence polynucleotides
comprising sequence regions that have substantial sequence identity
and can be homologously recombined in vitro or in vivo.
[0035] The terms cystathionine gamma synthase nucleic acid and
serine acetyl transferase nucleic acid used in this application
refer to all nucleic acids described herein or obtainable by the
methods described herein that, when utilized in the present
invention, alter the level of an organic sulfur compound.
[0036] In some instances, the enzymes of interest are natively
expressed in the plant. However, by transformation with
heterologous promoters, expression levels or patterns can be
altered. See, for example, Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.). and Innis et al. (1990) PCR Protocols: A
Guide to Methods and Applications (Academic Press, New York).
[0037] The nucleic acids can be combined with constitutive or
non-seed tissue-specific promoters for expression of the metabolite
of interest. Such constitutive promoters include, for example, the
core promoter of the Rsyn7 (copending U.S. patent application Ser.
No. 08/661,601), the core CaMV 35S promoter (Odell et al. (1985)
Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell
2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.
12:619-632 and Christensen et al. (1992) Plant Mol. Biol.
18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.
81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS
promoter (U.S. patent application Ser. No. 08/409,297), and the
like. Other constitutive promoters include, for example, U.S. Pat.
Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;
5,399,680; 5,268,463; and 5,608,142.
[0038] Non-seed tissue specific promoters include, for example
Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al.
(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol.
Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.
6(2):157-168; Rinehart et al. (1996) Plant Physiol.
112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.
112(2):525-535; Canevascini et al. (1996) Plant Physiol.
12(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. All of such
promoters can be modified, if necessary, for weak expression.
[0039] Leaf-preferred promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0040] Root-preferred promoters are known and can be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol. Biol. 20(2): 207-218 (soybean root-preferred glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-preferred control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-preferred promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also Bogusz et al. (1990) Plant Cell
2(7):633-641, where two root-preferred promoters isolated from
hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema
tomentosa are described. The promoters of these genes were linked
to a .beta.-glucuronidase reporter gene and introduced into both
the nonlegume Nicotiana tabacum and the legume Lotus corniculatus,
and in both instances root-preferred promoter activity was
preserved. Leach and Aoyagi (1991) describe their analysis of the
promoters of the highly expressed roIC and roID root-inducing genes
of Agrobacterium rhizogenes (see Plant Science (Limerick)
79(1):69-76). They concluded that enhancer and tissue-preferred DNA
determinants are dissociated in those promoters. Teeri et al.
(1989) used gene fusion to lacZ to show that the Agrobacterium
T-DNA gene encoding octopine synthase is especially active in the
epidermis of the root tip and that the TR2' gene is root specific
in the intact plant and stimulated by wounding in leaf tissue, an
especially desirable combination of characteristics for use with an
insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The
TR1' gene, fused to nptll (neomycin phosphotransferase 11) showed
similar characteristics. Additional root-preferred promoters
include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant
Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994)
Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876;
5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and
5,023,179. Tissue preferred and specific promoters also include the
tissue specific and tissue preferred promoters listed in PCT
application publication number WO 00/36124, Page 35, Table A, which
table is hereby incorporated by reference.
[0041] "Seed-specific" promoters, such as globulin 1, glutelin 2,
cruciferin, napin, B-conglycinin, phaseolin, as well as other seed
or endosperm specific promoters are not appropriate for use in the
present invention.
[0042] The promoter may be native or analogous or foreign or
heterologous to the plant host. Additionally, the promoter may be a
synthetic sequence. By foreign is intended that the transcriptional
initiation region is not found in the native plant into which the
transcriptional initiation region is introduced. Although both
monocot and dicot promoters are described herein, monocot promoters
are preferred for use in the present invention.
[0043] Expression cassettes will comprise a promoter linked to the
coding sequence or antisense sequence of the nucleotide of
interest. Such an expression cassette is generally provided with a
plurality of restriction sites for insertion of the sequence to be
under the transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
[0044] The transcriptional cassette will include in the 5'-to-3'
direction of transcription, a transcriptional and translational
initiation region, a DNA sequence of interest, and a
transcriptional and translational termination region functional in
plants. The termination region may be native with the
transcriptional initiation region, may be native with the DNA
sequence of interest, or may be derived from another source.
Convenient termination regions are available from the Ti-plasmid of
A. tumefaciens, such as the octopine synthase and nopaline synthase
termination regions. See also Guerineau et al. (1991) Mol. Gen.
Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et
al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell.
2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al.
(1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic
Acids Res. 15:9627-9639.
[0045] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0046] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell targeted for transformation.
Suitable methods of introducing nucleotide sequences into plant
cells and subsequent insertion into the plant genome include
microinjection (Crossway et al. (1986) Biotechniques 4:320-334),
electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA
83:5602-5606, Agrobacterium-mediated transformation (Townsend et
al., U.S. Pat. No. 5,563,055); direct gene transfer (Paszkowski et
al. (1984) EMBO J. 3:2717-2722), and ballistic particle
acceleration (see, for example, Sanford et al., U.S. Pat. No.
4,945,050; Tomes et al. (1995) "Direct DNA Transfer into Intact
Plant Cells via Microprojectile Bombardment," in Plant Cell,
Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and
Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)
Biotechnology 6:923-926). Also see Datta et al. (1990)
Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl.
Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)
Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855;
Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et
al. (1995) "Direct DNA Transfer into Intact Plant Cells via
Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)
(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize);
Fromm et al. (1990) Biotechnology 8:833-839 (maize); De Wet et al.
(1985) in The Experimental Manipulation of Ovule Tissues, ed.
Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler
et al. (1990) Plant Cell Reports 9:415-418; and Kaeppler et al.
(1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated
transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505
(electroporation); Li et al. (1993) Plant Cell Reports 12:250-255
and Christou and Ford (1995) Annals of Botany 75:407-413 (rice);
Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via
Agrobacterium tumefaciens); all of which are herein incorporated by
reference.
[0047] The biosynthesis of organic sulfur compounds can be altered
in accordance with the present invention in any monocot plant of
interest. Of particular interest are plants useful for human and
domestic animal food. Such plants include forages and seed crop
plants such as cereal crops. Of particular interest are plants
where the seed is produced in high amounts, or the seed or a seed
part is edible. Seeds of interest include the grain seeds such as
wheat, barley, rice, corn, rye, millet and sorghum. Especially
preferred plants are corn, wheat and rice.
[0048] The modified plant may be grown into plants in accordance
with conventional ways. See, for example, McCormick et al. (1986)
Plant Cell. Reports 5:81-84. These plants may then be grown, and
either pollinated with the same transformed strain or different
strains, and the resulting hybrid having the desired phenotypic
characteristic identified. Two or more generations may be grown to
ensure that the subject phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure the
desired phenotype or other property has been achieved.
[0049] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1
Transformation and Regeneration of Transgenic Maize Plants with
Cystathionine Gamma Synthase
[0050] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing cystathionine gamma synthase
nucleotide sequence (Accession No. AF007786) operably linked to a
ubiquitin promoter (U.S. Pat. Nos. 5,510,474 and 5,614,399) that
has been optimized for maize codon preference and a pin 11
terminator (An et. al. 1989), plus the selectable marker gene PAT
(Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to
the herbicide Bialaphos.
Transformation of Maize
[0051] Freshly isolated immature embryos of maize, about 10 days
after pollination (DAP), are cultured for 4-5 days before
transforming DNA is delivered via particle bombardment. The
preferred genotype for transformation is the highly transformable
genotype Hi-II (Armstrong, C. L., 1991, Development and
Availability of Germplasm with High Type II Culture Formation
Response, Maize Genetics Cooperation Newsletter, 65:92-93). An
F.sub.1 hybrid created by crossing with a Hi-II with an elite
inbred may also be used. After DNA delivery, the embryos are
cultured on medium containing toxic levels of herbicide. Only those
cells which receive the herbicide-resistance gene, and the linked
gene(s), grow on selective medium. Transgenic events so selected
are propagated and regenerated to whole plants, produce seed, and
transmit transgenes to progeny.
Particle Gun Terminology and Use
[0052] The PDS-1000 Biolistics particle bombardment device is used
to transform maize. The operation of this device is detailed in the
operating instructions available from the manufacturer (Bio-Rad
Laboratories, Hercules, Calif.).
[0053] The macrocarrier flight distance is fixed in the instrument
at 1/4'' (0.25''). While the rupture disk-macrocarrier gap distance
is adjustable, the device is operated at the factory recommended
distance of 1/8'' (0.125'').
Preparation of Particles
[0054] The transforming DNA is associated with either tungsten or
gold particles. Prior to association with the transforming DNA, the
tungsten particles are prepared essentially as described by Tomes
et al. (U.S. Pat. No. 5,990,387).
[0055] The preferred method utilizes gold particles. Gold particles
are prepared as follows. Sixty mg of 0.6.mu. gold particles
(Bio-Rad) are placed in 2.0 mL Sarstedt tube. The particles are
washed three times in absolute ethanol (100%). Each ethanol wash
involves adding one mL of absolute ethanol to the tube, sonicating
the tube briefly, vortexing the tube on high for one minute,
centrifuging the tube to pellet the particles and discarding the
supernatant. The particles are then washed two times in sterile
deionized water. Each wash involves adding one mL of sterile
deionized water to the tube, sonicating the tube briefly, vortexing
the tube on high for one minute, centrifuging the tube to pellet
the particles and discarding the supernatant. Following the ethanol
and water wash steps, one mL of sterile deionized water is added to
the tube and the tube is sonicated. Aliquots (250 .mu.L) of the
particle-containing suspension are removed to siliconized 1.5 mL
tubes and combined with 750 .mu.L sterile deionized water.
Association of Particles with Transforming DNA
[0056] The transforming DNA is associated with the prepared
tungsten or gold particles by precipitation in a solution
comprising CaCl.sub.2 and spermidine as follows. A tube containing
tungsten or gold particles prepared as described above is sonicated
for 3 seconds at setting 2.5 in a water bath probe, Branson
Sonicator #450 (Branson Ultrasonics Corp., Danbury Conn.). Ten
.mu.L plasmid DNA (1 .mu.g plasmid total) in TE buffer is added to
the tube and mixed for 5 seconds. Next, 100 .mu.L 2.5 M CaCl.sub.2
and 10 .mu.L 0.1 M spermidine are added. The tube is then shaken on
a vortexer for 10 minutes followed by centrifugation for 30 seconds
at 10,000 rpm. The supernatant is removed and discarded, and 500
.mu.L absolute ethanol is added. The tube is then sonicated at
setting 2.5 for 3 seconds, centrifuged for 30 seconds at 10,000 rpm
and the supernatant removed. To the tube, 105 .mu.L of absolute
ethanol is added. The tube is sonicated for 3 seconds at setting
2.5 before placing a 10 .mu.L aliquot onto the center of a
macrocarrier.
Preparation of Target Tissue
[0057] Ears of Hi-II or Hi-II X elite inbred are sampled in planta
to assess the developmental stage of the embryos. When immature
embryos first become opaque, about 9-12 days post-pollination, the
ears are harvested for embryo dissection. The embryos are
approximately 1.5-1.8 mm long from coleoptilar to coleorhizal end.
Immature embryos are the target tissue for transient and stable
transformation experiments.
[0058] The ears are surface sterilized in 50% (v/v) Clorox
bleach+0.5% (v/v) Micro detergent for 20 minutes, and then rinsed
twice with sterile water. The immature embryos are excised from the
caryopsis and placed embryo axis side down (scutellum side up) onto
transformation support medium.
[0059] Embryos are cultured on 560L medium for 4-5 days in darkness
at 28.degree. C. At this time, a small amount of incipient
embryogenic tissue can be observed at the coleorhizal end of the
scutellum, but there is no production of subculturable tissue.
Delivery of DNA
[0060] As preparation for bombardment, the 4 day pre-cultured
embryos are transferred to 561Y medium, which contains elevated
sucrose, and are incubated in darkness at 28.degree. C. for 4
hours. The embryos are arranged, 10 embryos per plate, in a 2 cm
target area. The embryos are angled with their coleorhizal end
pointing up toward the macrocarrier at approximately a 30.degree.
angle. This orientation of the cultured embryos enhances exposure
of the preferred cell targets to the path of particles propelled by
the particle gun.
[0061] Plates of embryos are bombarded at shelf 2 from the bottom
of the device, 650 PSI rupture disk, and a chamber vacuum of 28 mm
Hg.
[0062] The bombarded plates are incubated in darkness at 28.degree.
C. for two days. After the two-day bombardment recovery period, the
embryos are transferred to Petri dishes containing 560 R medium.
This latter medium is comprised of those components which typically
are used to initiate and promote embryogenic tissue from maize
embryos, and contains 2% sucrose, and 3 ppm bialaphos as a
selective agent. The plates are incubated in darkness at 28.degree.
C. for 4-6 weeks, or until growth of putatively transformed events
are observed. 560 R culture medium does not support the growth of
untransformed tissue derived from the bombarded embryos. Therefore,
only putatively transformed tissue, resistant to bialaphos as a
consequence of expressing the resistance transgene, are competent
to grow.
[0063] Putatively transformed events are identified first by their
growth under selective conditions and individually subcultured to
fresh 560 R medium for propagation. Samples of each event are
assayed for their transgenic nature by PCR reaction using primer
sets designed to specifically amplify sequences in the inserted
gene(s).
Regeneration of T.sub.0 Plants
[0064] Transformed, selection-resistant embryogenic tissue is
transferred to 288 J medium to initiate plant regeneration.
Following somatic embryo maturation (2-4 weeks), well-developed
somatic embryos are transferred to medium for germination (272 V)
and transferred to a lighted culture room. Approximately 7-10 days
later, developing plantlets are transferred to 272 V hormone-free
medium in tubes for 7-10 days until plantlets are well established.
Plants are then transferred to inserts in flats (equivalent to
2.5'' pot) containing potting soil and grown for 1 week in a growth
chamber, subsequently grown an additional 1-2 weeks in the
greenhouse, then transferred to 1.6 gallon pots and grown to
maturity. Plants are monitored and scored for altered cystathionine
gamma synthase and/or phenotype such as increased sulfur
compounds.
Media Recipes
[0065] Medium 288 J contains the following ingredients: 950.000 ml
of D-I H.sub.2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol;
5.000 ml of MS Vitamins Stock Solution (No. 36J); 1.000 ml of
Zeatin.5 mg/ml; 60.000 g of Sucrose; 3.000 g of Gelrite, which is
added after Q.S. to volume; 2.000 ml of IAA 0.5 mg/ml #; 1.000 ml
of 0.1 Mm ABA #; 3.000 ml of Bialaphos 1 mg/ml #; and 2.000 ml of
Agribio Carbenicillin 50 mg/ml #. Directions are: dissolve
ingredients in polished D-I H.sub.2O in sequence; adjust to pH 5.6;
Q.S. to volume with polished D-I H.sub.2O after adjusting pH;
sterilize and cool to 60.degree. C. Add 3.5 g/L of Gelrite for cell
biology. Ingredients designated with # are added after sterilizing
and cooling to temperature.
[0066] Medium 272 V contains the following ingredients: 950.000 ml
of D-I H.sub.2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol;
5.000 ml of MS Vitamins Stock Solution; 40.000 g of Sucrose; and
6.000 g of Bactoagar, which is added after Q.S. to volume.
Directions are: dissolve ingredients in polished D-I H.sub.2O in
sequence; adjust to pH 5.6; Q.S. to volume with polished D-I
H.sub.2O after adjusting pH; and sterilize and cool to 60.degree.
C.
[0067] Medium 560 L contains the following ingredients: 950.000 ml
of D-I Water, Filtered; 4.000 g of CHU (N6) Basal Salts (SIGMA
C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000.times.
SIGMA-1511); 1.250 ml of Thiamine.HCL 0.4 mg/ml; 30.000 g of
Sucrose; 4.000 ml of 2,4-D 0.5 mg/ml; 3.000 g of Gelrite, which is
added after Q.S. to volume; and 0.425 ml of Silver Nitrate 2 mg/ml
#. Directions are: dissolve ingredients in D-I H.sub.2O in
sequence; adjust to pH 5.8 with KOH; bring up to volume with D-I
H.sub.2O; sterilize and cool to room temp. Total volume (L)=1.00.
Ingredients designated with # are added after sterilizing and
cooling to temperature.
[0068] Medium 560 R contains the following ingredients: 950.000 ml
D-I Water, Filtered; 4.000 g of CHU (N6) Basal Salts (SIGMA
C-1416); 1.000 ml Eriksson's Vitamin Mix (1000.times. SIGMA-1511);
1.250 ml of Thiamine.HCL 0.4 mg/ml; 30.000 g Sucrose; 4.000 ml of
2,4-D 0.5 mg/ml; 3.000 g of Gelrite, which is added after Q.S. to
volume; 0.425 ml of Silver Nitrate 2 mg/ml #; and 3.000 ml of
Bialaphos 1 mg/ml #. Directions are: dissolve ingredients in D-I
H.sub.2O in sequence; adjust to pH 5.8 with KOH; bring up to volume
with D-I H.sub.2O; sterilize and cool to room temp. Total volume
(L)=1.00. Ingredients designated with # are added after sterilizing
and cooling to temperature.
[0069] Medium 561 Y contains the following ingredients: 950.000 ml
of D-I Water, Filtered; 4.000 g of CHU (N6) Basal Salts (SIGMA
C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000.times.
SIGMA-1511); 1.250 ml of Thiamine.HCL 0.4 mg/ml; 190.000 g of
Sucrose; 2.000 ml of 2,4-D 0.5 mg/ml; 2.880 g of L-Proline; 2.000 g
of Gelrite, which is added after Q.S. to volume; and 4.250 ml of
Silver Nitrate 2 mg/ml #. Directions are: dissolve ingredients in
D-I H.sub.2O in sequence; adjust to pH 5.8 with KOH; bring up to
volume with D-I H.sub.2O; sterilize and cool to room temp.
Autoclave less time because of increased sucrose. Total volume
(L)=1.00. Ingredients designated with # are added after sterilizing
and cooling to temperature.
Example 2
Agrobacterium-Mediated Transformation of Maize with Cystathionine
Gamma Synthase
[0070] For Agrobacterium-mediated transformation of maize with a
cystathionine gamma synthase nucleotide sequence, a maize cDNA for
cystathionine gamma synthase (Accession No. AF007786) is fused to a
maize optimized ubiquitin promoter (U.S. Pat. Nos. 5,510,474 and
5,614,399) that has been optimized for maize codon preference and a
pin 11 terminator sequence (An, et al., 1989). The cystathionine
gamma synthase cassette, also containing a CaMV35S-bialaphos
selectable marker element, is cloned into a binary vector and
introduced into Agrobacterium.
Transformation of Maize Mediated by Agrobacterium
[0071] Freshly isolated immature embryos of maize, about 10 days
after pollination (DAP), are incubated with the Agrobacterium. The
preferred genotype for transformation is the highly transformable
genotype Hi-II (Armstrong, C. L., 1991, Development and
Availability of Germplasm with High Type II Culture Formation
Response, Maize Genetics Cooperation Newsletter, 65:92-93). An
F.sub.1 hybrid created by crossing with an Hi-II with an elite
inbred may also be used. After Agrobacterium treatment of immature
embryos, the embryos are cultured on medium containing toxic levels
of herbicide. Only those cells which receive the
herbicide-resistance gene, and the linked gene(s), grow on
selective medium. Transgenic events so selected are propagated and
regenerated to whole plants, produce seed, and transmit transgenes
to progeny.
Preparation of Agrobacterium
[0072] The engineered Agrobacterium tumefaciens LBA4404 is
constructed as per U.S. Pat. No. 5,591,616 to contain the linked
gene(s) and the selectable marker gene. Typically either BAR
(D'Halluin et al. (1992) Methods Enzymol. 216:415-426) or PAT
(Wohlleben et al. (1988) Gene 70:25-37) may be used.
[0073] To use the engineered vector in plant transformation, a
master plate of single bacterial colonies is first prepared by
inoculating the bacteria on minimal AB medium and then incubating
the bacteria plate inverted at 28.degree. C. in darkness for about
3 days. A working plate is then prepared by selecting a single
colony from the plate of minimal A medium and streaking it across a
plate of YP medium. The YP-medium bacterial plate is then incubated
inverted at 28.degree. C. in darkness for 1-2 days.
[0074] Agrobacterium for plant transfection and co-cultivation is
prepared 1 day prior to transformation. Into 30 ml of minimal A
medium in a flask is placed 50 .mu.g/ml spectinomycin (or
appropriate bacterial antibiotic depending on marker in
co-integrate), 100 .mu.M acetosyringone, and about a 1/8 loopful of
Agrobacterium from a 1 to 2-day-old working plate. The
Agrobacterium is then grown at 28.degree. C. at 200 rpm in darkness
overnight (about 14 hours). In mid-log phase, the Agrobacterium is
harvested and resuspended at 3 to 5.times.10.sup.8 CFU/ml in 561Q
medium+100 .mu.M acetosyringone using standard microbial techniques
and standard curves.
Immature Embryo Preparation
[0075] Nine to ten days after controlled pollination of a corn
plant, developing immature embryos are opaque and 1-1.5 mm long and
are the appropriate size for Agro-infection. The husked ears are
sterilized in 50% commercial bleach and 1 drop Tween for 30
minutes, and then rinsed twice with sterile water. The immature
embryos are aseptically removed from the caryopsis and placed into
2 ml of sterile holding solution comprising of 561Q+100 .mu.M
acetosyringone.
Agrobacterium Infection and Co-Cultivation of Embryos
[0076] Holding solution is decanted from excised immature embryos
and replaced with prepared Agrobacterium. Following gentle mixing
and incubation for about 5 minutes, the Agrobacterium is decanted
from the immature embryos. Immature embryos are then moved to a
plate of 562P medium, scutellum surface upwards, and incubated at
20.degree. C. for 3 days in darkness followed by incubation at
28.degree. C. for 3 days in darkness on medium 562P+100 mg/ml
carbenecillin (see U.S. Pat. No. 5,981,840).
Selection of Transgenic Events
[0077] Following incubation, the immature embryos are transferred
to 5630 medium for selection of events. The transforming DNA
possesses a herbicide-resistance gene, in this example the PAT
gene, which confers resistance to bialaphos. At 10- to 14-day
intervals, embryos are transferred to 5630 medium. Actively growing
putative transgenic embryogenic tissue are visible in 6-8
weeks.
Regeneration of T.sub.0 Plants
[0078] Transgenic embryogenic tissue is transferred to 288W medium
and incubated at 28.degree. C. in darkness until somatic embryos
matured, or about 10 to 18 days. Individual matured somatic embryos
with well-defined scutellum and coleoptile are transferred to 272
embryo germination medium and incubated at 28.degree. C. in the
light. After shoots and roots emerge, individual plants are potted
in soil and hardened-off using typical horticultural methods.
Confirmation of Transformation
[0079] Putative transgenic events are subjected to analysis to
confirm their transgenic nature.
[0080] Events are tested for the presence of the cystathionine
gamma synthase by PCR amplification. Additionally, T.sub.0 plants
are painted with bialaphos herbicide. The subsequent lack of a
herbicide-injury lesion indicates the presence and action of the
herbicide resistance gene. The plants are monitored and scored for
altered cystathionine gamma synthase expression and/or phenotype
such as increased organic sulfur compounds.
Media Recipes
[0081] Medium 561 Q contains the following ingredients: 950.000 ml
of D-I Water, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma
C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000.times.
Sigma-1511); 1.250 ml of Thiamine.HCL.4 mg/ml; 3.000 ml of 2,4-D
0.5 mg/ml (No. 2A); 0.690 g of L-proline; 68.500 g of Sucrose; and
36.000 g of Glucose. Directions are: dissolve ingredients in
polished D-I H.sub.2O in sequence; adjust pH to 5.2 w/KOH; Q.S. to
volume with polished D-I H.sub.2O after adjusting pH; and filter
sterilize (do not autoclave).
[0082] Medium 562 P contains the following ingredients: 950.000 ml
of D-I Water, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma
C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000.times.
Sigma-1511); 1.250 ml of Thiamine.HCL.4 mg/ml; 4.000 ml of 2,4-D
0.5 mg/ml; 0.690 g of L-proline; 30.000 g of Sucrose; 3.000 g of
Gelrite, which is added after Q.S. to volume; 0.425 ml of Silver
Nitrate 2 mg/ml #; and 1.000 ml of Aceto Syringone 100 mM #.
Directions are: dissolve ingredients in polished D-I H.sub.2O in
sequence; adjust pH to 5.8 w/KOH; Q.S. to volume with polished D-I
H.sub.2O after adjusting pH; and sterilize and cool to 60.degree.
C. Ingredients designated with a # are added after sterilizing and
cooling to temperature.
[0083] Medium 563 O contains the following ingredients: 950.000 ml
of D-I Water, Filtered; 4.000 g of Chu (N6) Basal Salts (Sigma
C-1416); 1.000 ml of Eriksson's Vitamin Mix (1000.times.
Sigma-1511); 1.250 ml of Thiamine.HCL.4 mg/ml; 30.000 g of Sucrose;
3.000 ml of 2,4-D 0.5 mg/ml (No. 2A); 0.690 g of L-proline; 0.500 g
of Mes Buffer; 8.000 g of Agar (Sigma A-7049, Purified), which is
added after Q.S. to volume; 0.425 ml of Silver Nitrate 2 mg/ml #;
3.000 ml of Bialaphos 1 mg/ml #; and 2.000 ml of Agribio
Carbenicillin 50 mg/ml #. Directions are: dissolve ingredients in
polished D-I H.sub.2O in sequence; adjust to pH 5.8 w/koh; Q.S. to
volume with polished D-I H.sub.2O after adjusting pH; sterilize and
cool to 60.degree. C. Ingredients designated with a # are added
after sterilizing and cooling to temperature.
[0084] Medium 288 W contains the following ingredients: 950.000 ml
of D-I H.sub.2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol;
5.000 ml of MS Vitamins Stock Solution (No. 36J); 1.000 ml of
Zeatin.5 mg/ml; 60.000 g of Sucrose; 8.000 g of Agar (Sigma A-7049,
Purified), which is added after Q.S. to volume; 2.000 ml of IAA 0.5
mg/ml #; 1.000 ml of 0.1 Mm ABA #; 3.000 ml of Bialaphos 1 mg/ml #;
and 2.000 ml of Agribio Carbenicillin 50 mg/ml #. Directions are:
dissolve ingredients in polished D-I H.sub.2O in sequence; adjust
to pH 5.6; Q.S. to volume with polished D-I H.sub.2O after
adjusting pH; sterilize and cool to 60.degree. C. Add 3.5 g/L of
Gelrite for cell biology. Ingredients designated with a # are added
after sterilizing and cooling to temperature.
[0085] Medium 272 contains the following ingredients: 950.000 ml of
D-I H2O; 4.300 g of MS Salts; 0.100 g of Myo-Inositol; 5.000 of MS
Vitamins Stock Solution; 40.000 g of Sucrose; and 1.500 g of
Gelrite, which is added after Q.S. to volume. Directions are:
dissolve ingredients in polished D-I H2O in sequence; adjust to pH
5.6; Q.S. to volume with polished D-I H2O after adjusting pH; and
sterilize and cool to 60.degree. C.
[0086] Medium minimal A contains the following ingredients: 950.000
ml of D-1 H.sub.2O; 10.500 g of potassium phosphate dibasic K2HPO4;
4.500 g of potassium phosphate monobasic KH2PO4; 1.000 g of
ammonium sulfate; 0.500 g of sodium citrate dihydrate; 10.000 ml of
sucrose 20% solution #; and 1.000 ml of 1M magnesium sulfate #.
Directions are: dissolve ingredients in polished D-I H2O in
sequence; Q.S. to volume with D-I H2O; sterilize and cool to
60.degree. C. Ingredients designated with a # are added after
sterilizing and cooling to temperature.
[0087] Medium minimal AB contains the following ingredients:
850.000 ml of D-I H.sub.2O; 50.000 ml of stock solution 800A; 9 g
of Phytagar which is added after Q.S. to volume; 50.000 ml of stock
solution 800B #; 5.000 g of glucose #; and 2.000 ml of
spectinomycin 50/mg/ml stock #. Directions are: dissolve
ingredients in polished D-I H2O in sequence; Q.S. to volume with
polished D-I H2O less 100 ml per liter; sterilize and cool to
60.degree. C. Ingredients designated with a # are added after
sterilizing and cooling to temperature. Stock solution 800A
contains the following ingredients: 950.000 ml of D-I H2O; 60.000 g
of potassium phosphate dibasic K2HPO4; and 20.000 g of sodium phos.
monobasic, hydrous. Directions are: dissolve ingredients in
polished D-I H2O in sequence; adjust pH to 7.0 w/koh; Q.S. to
volume with polished D-I H2O after adjusting pH; and sterilize and
cool to 60.degree. C. Stock solution 800B contains the following
ingredients: 950.000 ml of D-I H2O; 20.000 g of ammonium chloride;
6.000 g of magnesium sulfate 7-H.sub.2O, MgSO4, 7H2O; 3.000 g of
potassium chloride; 0.200 g of calcium chloride (anhydrate); and
0.050 g of ferrous sulfate 7-hydrate. Directions are: dissolve
ingredients in polished D-I H2O in sequence; Q.S. to volume with
polished D-I H.sub.2O; and sterilize and cool to 60.degree. C.
[0088] Medium minimal YP contains the following ingredients:
950.000 ml of D-I H.sub.2O; 5.000 g of yeast extract (Difco);
10.000 g of peptone (Difco); 5.000 g of sodium chloride; 15.000 g
of bacto-agar, which is added after Q.S. to volume; and 1.000 ml of
spectinomycin 50 mg/ml stock #. Directions are: dissolve
ingredients in polished D-I H2O in sequence; adjust pH to 6.8
w/koh; Q.S. to volume with polished D-I H2O after adjusting pH;
sterilize and cool to 60.degree. C. Ingredients designated with a #
are added after sterilizing and cooling to temperature.
Results
[0089] Following two generations of selfing, plants homozygous for
the cystathionine gamma synthase construct and null segregates
derived from more than 40 events are analyzed. As can be seen in
FIG. 2, forty seeds from each of three events homozygous for
cystathionine gamma synthase (6337, 6326, 6311; shown by shaded
bars on FIGS. 2 and 3) and null segregants (C1-C3; shown by open
bars on FIGS. 2 and 3) are analyzed for total sulfur content of
amino acids. (Beckman Instruments 6300 analyzer--amino acids
detected with ninhydrin.) Events homozygous for cystathionine gamma
synthase show a statistically significant increase in levels of
cysteine and methionine over the null segregates. The cysteine and
methionine levels in the homozygous events greatly surpass any
increase previously achieved through enzymatic modification with a
38% increase in cysteine on a dry weight basis and a 49% increased
in methionine on a dry weight basis (FIG. 2).
[0090] As can be seen in FIG. 3, when expressed on a mol percent
basis, the data shows that the concentration of cysteine has
increased by 9% and methionine has increased by 20%, each of which
is statistically significant at a p value of less than 0.05. Over
expression of cystathionine gamma synthase in transgenic lines was
also confirmed by western blot analysis. Thus, Applicants show that
constitutive expression of cystathionine gamma synthase leads to
substantial and unexpected increases in total seed cysteine and
methionine levels.
Example 3
Transformation and Regeneration of Transgenic Maize Callus with
Serine Acetyl Transferase
[0091] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing serine acetyl transferase
nucleotide sequence (Seq. ID No. 1) operably linked to a ubiquitin
promoter (U.S. Pat. Nos. 5,510,474 and 5,614,399) that has been
optimized for maize codon preference and a pin II terminator (An
et. al. 1989), plus a plasmid containing the selectable marker gene
PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance
to the herbicide Bialaphos. Transformation is performed according
to the procedure described in Example 1.
Example 4
Agrobacterium-Mediated Transformation of Maize with Serine Acetyl
Transferase
[0092] For Agrobacterium-mediated transformation of maize with a
serine acetyl transferase nucleotide sequence, a maize cDNA for
serine acetyl transferase (Sequence ID No. 1) is fused to a
ubiquitin promoter sequence (U.S. Pat. Nos. 5,510,474 and
5,617,399) that has been optimized for maize codon preference and a
pin 11 terminator sequence (An, et al., 1989). The serine acetyl
transferase cassette, also contains a CaMV35S-bialaphos selectable
marker element, is cloned into a binary vector and introduced into
Agrobacterium. Transformation is performed according to the
procedure described in Example 2.
Example 5
Agrobacterium-Mediated Transformation of Maize with Serine Acetyl
Transferase and ATP Sulfurylase
[0093] Transgenic plants are constructed by Applicants according to
the method described in Example 4, but with an expression cassette
containing both the serine acetyl transferase sequence described in
Example 4 and an ATP Sulfurylase sequence (Sequence ID No. 3)
linked to a ubiquitin promoter sequence (U.S. Pat. Nos. 5,510,474
and 5,617,399) that has been optimized for maize codon preference
and a Pin II terminator sequence (An, et al., 1989).
Results
[0094] More than twenty events producing T1 seed are analyzed for
overexpression of both genes by western blot analysis. Twenty to
forty seeds from each of two events show strong expression of both
ATP Sulfurylase and serine acetyl transferase, and two events
showing weak or undetectable expression are analyzed for total
sulfur amino acid content (Beckman Instruments 6300 analyzer--amino
acids detected with ninhydrin). These results are shown in FIGS. 4
and 5. Samples 3770 and 3768 (represented by the shaded bars in
FIGS. 4 and 5) showed strong expression of ATP Sulfurylase and
serine acetyl transferase, while samples 3778 and 3753 (represented
by the open bars in FIGS. 4 and 5) showed weak expression of ATP
Sulfurylase and serine acetyl transferase. Segregating seed from
event 3768 is propagated, selfed, and the plants are analyzed for
the presence of the construct by their resistance to herbicide. T2
(second generation) seed was harvested from both resistant and
susceptible plants. Sulfur amino acids were analyzed for total
sulfur amino acid content using a Beckman Instruments 6300
analyzer, with the amino acids detected by ninhydrin. Results are
shown in FIGS. 6 and 7 with the herbicide resistant lines (+
construct) represented by shaded bars and the herbicide susceptible
lines (- construct) represented by open bars.
[0095] The cysteine and methionine levels in the plants with serine
acetyl transferase and ATP Sulfurylase greatly surpassed any
increase previously achieved through enzymatic modification with a
20% average increase in cysteine on a dry weight basis and a 97%
average increase in methionine on a dry weight basis (FIG. 6). As
can be seen in FIG. 7, when expressed on a mol percent basis, the
data shows that the concentration of cysteine has increased by an
average of 19% and methionine has increased by an average of 63%.
Although the construct used by Applicants contains both serine
acetyl transferase and ATP Sulfurylase, Applicant believes that the
increase in the cysteine and methionine levels of the transformed
plants is due to the serine acetyl transferase rather than the ATP
Sulfurylase. Thus, the preferred embodiment is the construct taught
herein that comprises only the serine acetyl transferase nucleic
acid and not the ATP Sulfurylase nucleic acid. However, the data
demonstrates that a construct with both the serine acetyl
transferase nucleic acid and the ATP Sulfurylase nucleic acid may
be used to modulate the biosynthesis of at least one organic sulfur
compound. Similarly, only the ATP Sulfurylase nucleic acid and not
the serine acetyl transferase nucleic may also be used to modulate
the biosynthesis of at least one organic sulfur compound. Such
constructs, and plants with such constructs, may be produced by the
methods taught in the Examples described herein.
[0096] Although Applicant has taught examples using specific
enzymes that it believes to be preferred for use in the present
invention, Applicant has more generally taught that, to increase
sulfur amino acids and organic sulfur compounds via enzymatic
modification in the seed of a monocot plant, it is sufficient to
express such enzymes in the non-seed tissue of the monocot plant.
The plant will then translocate these sulfur amino acids and
organic sulfur compounds to the seed, where the seed, without any
genetic modification, will accumulate such amino acids and
compounds. Applicant reasonably believes that any one or more of
the enzymes along the sulfate/serine to cysteine pathways (FIG. 1)
will function in the manner as taught herein. In such case, a
construct may be produced in the manner described herein but using
such enzyme or enzymes. One or more enzymes in the sulfate to
cysteine and/or the serine to cysteine pathways are referred to
herein as "sulfur assimilating enzymes." Such term also includes
additional enzymes that may activate or deactivate such enzymes,
such as kinases that activate or inactivate such enzymes by
phosphorylation.
[0097] In summary, Applicant has shown that constitutive or
non-seed expression of one or more enzymes in the cysteine
biosynthetic pathway leads to substantial and unexpected increases
in total seed cysteine and methionine levels. This increase in
cysteine and methionine can be utilized as a sulfur source for the
production of other organic sulfur compounds. Subsequent production
of the other organic sulfur compounds from this increased sulfur
source may occur in either the seed, leaf, root, stem or other
plant tissue.
[0098] All publications, patents and patent applications mentioned
in the specification are indicative of the level of those skilled
in the art to which this invention pertains. All publications,
patents and patent applications are herein incorporated by
reference to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated by reference.
[0099] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
4 1 927 DNA Zea mays CDS (1)...(925) 1 atg acg gcc ggg cag ctt ctg
cgc acc gag cca tca gcc cag ccc cag 48 Met Thr Ala Gly Gln Leu Leu
Arg Thr Glu Pro Ser Ala Gln Pro Gln 1 5 10 15 cgg gtg cgc cac agc
acc ccg ccg gcg gca ctc caa gca gac atc gtg 96 Arg Val Arg His Ser
Thr Pro Pro Ala Ala Leu Gln Ala Asp Ile Val 20 25 30 ccg tcg tac
ccg ccg ccc gag tcg gac ggt gac gag tcg tgg gtc tgg 144 Pro Ser Tyr
Pro Pro Pro Glu Ser Asp Gly Asp Glu Ser Trp Val Trp 35 40 45 tcc
cag atc aag gcg gag gcg cgg cgc gac gcg gac gcg gag ccg gcg 192 Ser
Gln Ile Lys Ala Glu Ala Arg Arg Asp Ala Asp Ala Glu Pro Ala 50 55
60 ctg gcc tcc ttc ctc tac gcg acg gtg ctg tcg cac gcg tcc ctg gac
240 Leu Ala Ser Phe Leu Tyr Ala Thr Val Leu Ser His Ala Ser Leu Asp
65 70 75 80 cgg tcc ctg gcc ttc cac ctg gcc aac aag ctg tgc tcc tcc
acg ctg 288 Arg Ser Leu Ala Phe His Leu Ala Asn Lys Leu Cys Ser Ser
Thr Leu 85 90 95 ctg tcg acg ctc tct aac gac ctc ttc gtg gcg tcg
ctc gcg gag cac 336 Leu Ser Thr Leu Ser Asn Asp Leu Phe Val Ala Ser
Leu Ala Glu His 100 105 110 ccg tcg tcc gcg cgg cgg cgg tgg cga cct
gat cgc cgc gcg gtc gcg 384 Pro Ser Ser Ala Arg Arg Arg Trp Arg Pro
Asp Arg Arg Ala Val Ala 115 120 125 gga ccc ggc tgc gcg ggc ttc gcg
cac tgc ctc ctc aac tac aag ggg 432 Gly Pro Gly Cys Ala Gly Phe Ala
His Cys Leu Leu Asn Tyr Lys Gly 130 135 140 ttc ctg gcc gtg cag gcg
cac cgc gtg gcg cac gtg ctg tgg gcg cag 480 Phe Leu Ala Val Gln Ala
His Arg Val Ala His Val Leu Trp Ala Gln 145 150 155 160 ggc cgg cgc
gcg ctg gcg ctg gcg ctc cag tcc cgc gtc gcc gag gtc 528 Gly Arg Arg
Ala Leu Ala Leu Ala Leu Gln Ser Arg Val Ala Glu Val 165 170 175 ttc
gcc gtg gac atc cac ccg gcc gcc acc gtc ggc agg ggc atc ctg 576 Phe
Ala Val Asp Ile His Pro Ala Ala Thr Val Gly Arg Gly Ile Leu 180 185
190 ctc gac cac gcc acg ggc gtc gtc gtc ggg gag acg gcc gtc gtg ggc
624 Leu Asp His Ala Thr Gly Val Val Val Gly Glu Thr Ala Val Val Gly
195 200 205 gac aac gtc tcc ata ctc cac cac gtg acg ttg gcg gca ccg
gca agg 672 Asp Asn Val Ser Ile Leu His His Val Thr Leu Ala Ala Pro
Ala Arg 210 215 220 cgt tgg cga ccg gca ccc caa gat cgg gac ggc gtg
ctc atc ggc gcc 720 Arg Trp Arg Pro Ala Pro Gln Asp Arg Asp Gly Val
Leu Ile Gly Ala 225 230 235 240 ggc gcg acc gtc ctc gga aac gtc agg
atc ggc gcc ggc gcc aag gtc 768 Gly Ala Thr Val Leu Gly Asn Val Arg
Ile Gly Ala Gly Ala Lys Val 245 250 255 ggc gcc ggg tcc gtc gtg ctc
atc gac gtg ccg ccc agg agc acc gcc 816 Gly Ala Gly Ser Val Val Leu
Ile Asp Val Pro Pro Arg Ser Thr Ala 260 265 270 gtg ggg aac ccc gcc
agg ctg atc ggc ggg aag aag ggc gag gag gtg 864 Val Gly Asn Pro Ala
Arg Leu Ile Gly Gly Lys Lys Gly Glu Glu Val 275 280 285 atg ccg ggg
gag tcc atg gac cac acc tcc ttc ata cag cag tgg tcg 912 Met Pro Gly
Glu Ser Met Asp His Thr Ser Phe Ile Gln Gln Trp Ser 290 295 300 gac
tac atc att t ga 927 Asp Tyr Ile Ile 305 2 308 PRT Zea mays 2 Met
Thr Ala Gly Gln Leu Leu Arg Thr Glu Pro Ser Ala Gln Pro Gln 1 5 10
15 Arg Val Arg His Ser Thr Pro Pro Ala Ala Leu Gln Ala Asp Ile Val
20 25 30 Pro Ser Tyr Pro Pro Pro Glu Ser Asp Gly Asp Glu Ser Trp
Val Trp 35 40 45 Ser Gln Ile Lys Ala Glu Ala Arg Arg Asp Ala Asp
Ala Glu Pro Ala 50 55 60 Leu Ala Ser Phe Leu Tyr Ala Thr Val Leu
Ser His Ala Ser Leu Asp 65 70 75 80 Arg Ser Leu Ala Phe His Leu Ala
Asn Lys Leu Cys Ser Ser Thr Leu 85 90 95 Leu Ser Thr Leu Ser Asn
Asp Leu Phe Val Ala Ser Leu Ala Glu His 100 105 110 Pro Ser Ser Ala
Arg Arg Arg Trp Arg Pro Asp Arg Arg Ala Val Ala 115 120 125 Gly Pro
Gly Cys Ala Gly Phe Ala His Cys Leu Leu Asn Tyr Lys Gly 130 135 140
Phe Leu Ala Val Gln Ala His Arg Val Ala His Val Leu Trp Ala Gln 145
150 155 160 Gly Arg Arg Ala Leu Ala Leu Ala Leu Gln Ser Arg Val Ala
Glu Val 165 170 175 Phe Ala Val Asp Ile His Pro Ala Ala Thr Val Gly
Arg Gly Ile Leu 180 185 190 Leu Asp His Ala Thr Gly Val Val Val Gly
Glu Thr Ala Val Val Gly 195 200 205 Asp Asn Val Ser Ile Leu His His
Val Thr Leu Ala Ala Pro Ala Arg 210 215 220 Arg Trp Arg Pro Ala Pro
Gln Asp Arg Asp Gly Val Leu Ile Gly Ala 225 230 235 240 Gly Ala Thr
Val Leu Gly Asn Val Arg Ile Gly Ala Gly Ala Lys Val 245 250 255 Gly
Ala Gly Ser Val Val Leu Ile Asp Val Pro Pro Arg Ser Thr Ala 260 265
270 Val Gly Asn Pro Ala Arg Leu Ile Gly Gly Lys Lys Gly Glu Glu Val
275 280 285 Met Pro Gly Glu Ser Met Asp His Thr Ser Phe Ile Gln Gln
Trp Ser 290 295 300 Asp Tyr Ile Ile 305 3 1470 DNA Zea mays CDS
(1)...(1468) 3 atg gcg aca cag gcc gct ttc ctc gca ggg ttc tcg cag
ctc gcc gcg 48 Met Ala Thr Gln Ala Ala Phe Leu Ala Gly Phe Ser Gln
Leu Ala Ala 1 5 10 15 cag ccg ggc cgc gac cgc gcc gtg gcg gtg gcg
gtg gcg ccg gcg ccg 96 Gln Pro Gly Arg Asp Arg Ala Val Ala Val Ala
Val Ala Pro Ala Pro 20 25 30 ggc ccg gcc cgg gtg gcc gtt gcg gcg
gtg ggt agc gcc aag ttg ggc 144 Gly Pro Ala Arg Val Ala Val Ala Ala
Val Gly Ser Ala Lys Leu Gly 35 40 45 gtg aag gcg ggg acg tcc agg
acc gcg gcg gtg gcg cgc ctc ggg gtg 192 Val Lys Ala Gly Thr Ser Arg
Thr Ala Ala Val Ala Arg Leu Gly Val 50 55 60 cgg tgc cgg gcc agc
ctg atc gag ccc gac ggc ggg cgg ctg gtg gac 240 Arg Cys Arg Ala Ser
Leu Ile Glu Pro Asp Gly Gly Arg Leu Val Asp 65 70 75 80 ctg gtg gcg
ccc gag gag ggc ggg cgg cgc gcg gcg ctg cgg cgg gag 288 Leu Val Ala
Pro Glu Glu Gly Gly Arg Arg Ala Ala Leu Arg Arg Glu 85 90 95 gcg
gcg gag ctg ccg cac cgg ctg cgc ttg ggc cgc gtc gac aag gaa 336 Ala
Ala Glu Leu Pro His Arg Leu Arg Leu Gly Arg Val Asp Lys Glu 100 105
110 tgg gtc cac gtc ctc agc gaa ggg tgg gcg agc ccg ctg caa ggg ttc
384 Trp Val His Val Leu Ser Glu Gly Trp Ala Ser Pro Leu Gln Gly Phe
115 120 125 atg cgc gag cat gag ttc ctc caa gca ctt cat ttc aat gcc
atc cgc 432 Met Arg Glu His Glu Phe Leu Gln Ala Leu His Phe Asn Ala
Ile Arg 130 135 140 ggc cag gat ggc agg atg gtc aac atg tcc gtc ccc
atc gtg ctc tct 480 Gly Gln Asp Gly Arg Met Val Asn Met Ser Val Pro
Ile Val Leu Ser 145 150 155 160 gtc ggg gac gca cag cga agg gcc atc
cag gcc gac ggc gcc acg cgc 528 Val Gly Asp Ala Gln Arg Arg Ala Ile
Gln Ala Asp Gly Ala Thr Arg 165 170 175 gtc gcg ctc gtt gac gag cgc
gac cgc ccc atc gcc gtc ctc agc gac 576 Val Ala Leu Val Asp Glu Arg
Asp Arg Pro Ile Ala Val Leu Ser Asp 180 185 190 att gag atc tat aag
cat aat aag gaa gaa agg gta gca cgg aca tgg 624 Ile Glu Ile Tyr Lys
His Asn Lys Glu Glu Arg Val Ala Arg Thr Trp 195 200 205 ggg aca act
gca cct gga tta cct tat gtc gag gag gca att acc aat 672 Gly Thr Thr
Ala Pro Gly Leu Pro Tyr Val Glu Glu Ala Ile Thr Asn 210 215 220 gct
ggt gac tgg ttg gtt ggt ggg gac ttg gag gtt ata gaa cca atc 720 Ala
Gly Asp Trp Leu Val Gly Gly Asp Leu Glu Val Ile Glu Pro Ile 225 230
235 240 aag tac aac gat ggt cta gat cag tat cgc ctg tct cca gca cag
ctg 768 Lys Tyr Asn Asp Gly Leu Asp Gln Tyr Arg Leu Ser Pro Ala Gln
Leu 245 250 255 cgt gaa gag ttt gcc agg cgc aat gct gat gca gta ttt
gcc ttt cag 816 Arg Glu Glu Phe Ala Arg Arg Asn Ala Asp Ala Val Phe
Ala Phe Gln 260 265 270 ctt cgc aat cct gta cac aat ggg cat gct ctt
ctt atg acc gac aca 864 Leu Arg Asn Pro Val His Asn Gly His Ala Leu
Leu Met Thr Asp Thr 275 280 285 cgc aaa cgt ctc ctt gag atg ggt tat
aaa aac cct gtt ctt ctg ctc 912 Arg Lys Arg Leu Leu Glu Met Gly Tyr
Lys Asn Pro Val Leu Leu Leu 290 295 300 cat cca ctg gga gga ttc aca
aaa gca gat gat gtg cct ctt agt tgg 960 His Pro Leu Gly Gly Phe Thr
Lys Ala Asp Asp Val Pro Leu Ser Trp 305 310 315 320 aga atg aag caa
cat gag aag gtt ctt gag gaa ggt gtc ctc aac cca 1008 Arg Met Lys
Gln His Glu Lys Val Leu Glu Glu Gly Val Leu Asn Pro 325 330 335 gaa
tca act gtt gtt gcg atc ttt ccc tct cca atg cat tat gct ggg 1056
Glu Ser Thr Val Val Ala Ile Phe Pro Ser Pro Met His Tyr Ala Gly 340
345 350 cca act gag gtg cag tgg cat gct aag gct cgt att aat gct ggt
gca 1104 Pro Thr Glu Val Gln Trp His Ala Lys Ala Arg Ile Asn Ala
Gly Ala 355 360 365 aat ttc tat att gtt gga agg gat cct gct ggt atg
agc cat ccc acg 1152 Asn Phe Tyr Ile Val Gly Arg Asp Pro Ala Gly
Met Ser His Pro Thr 370 375 380 gag aaa agg gac ctc tat gat gct gac
cac ggg aag aag gtt ttg agc 1200 Glu Lys Arg Asp Leu Tyr Asp Ala
Asp His Gly Lys Lys Val Leu Ser 385 390 395 400 atg gct cct ggc ctc
gag agg ctc aac atc ctt cct ttc aag gtg gct 1248 Met Ala Pro Gly
Leu Glu Arg Leu Asn Ile Leu Pro Phe Lys Val Ala 405 410 415 gca tat
gac aca aag caa aag aaa atg gat ttc ttc gat cca tca agg 1296 Ala
Tyr Asp Thr Lys Gln Lys Lys Met Asp Phe Phe Asp Pro Ser Arg 420 425
430 aaa gat gat ttc ctc ttc atc tct ggc aca aag atg cgc act ctt gcc
1344 Lys Asp Asp Phe Leu Phe Ile Ser Gly Thr Lys Met Arg Thr Leu
Ala 435 440 445 aag aac cgc gag agt ccc ccg gat ggt ttt atg tgc ccg
ggt ggc tgg 1392 Lys Asn Arg Glu Ser Pro Pro Asp Gly Phe Met Cys
Pro Gly Gly Trp 450 455 460 aaa gtg ctc gtt gaa tac tat gac agc ttg
gtg cca tcc gag ggc agc 1440 Lys Val Leu Val Glu Tyr Tyr Asp Ser
Leu Val Pro Ser Glu Gly Ser 465 470 475 480 agc aag ctg cgc gag cca
gtt gca gcc t ga 1470 Ser Lys Leu Arg Glu Pro Val Ala Ala 485 4 489
PRT Zea mays 4 Met Ala Thr Gln Ala Ala Phe Leu Ala Gly Phe Ser Gln
Leu Ala Ala 1 5 10 15 Gln Pro Gly Arg Asp Arg Ala Val Ala Val Ala
Val Ala Pro Ala Pro 20 25 30 Gly Pro Ala Arg Val Ala Val Ala Ala
Val Gly Ser Ala Lys Leu Gly 35 40 45 Val Lys Ala Gly Thr Ser Arg
Thr Ala Ala Val Ala Arg Leu Gly Val 50 55 60 Arg Cys Arg Ala Ser
Leu Ile Glu Pro Asp Gly Gly Arg Leu Val Asp 65 70 75 80 Leu Val Ala
Pro Glu Glu Gly Gly Arg Arg Ala Ala Leu Arg Arg Glu 85 90 95 Ala
Ala Glu Leu Pro His Arg Leu Arg Leu Gly Arg Val Asp Lys Glu 100 105
110 Trp Val His Val Leu Ser Glu Gly Trp Ala Ser Pro Leu Gln Gly Phe
115 120 125 Met Arg Glu His Glu Phe Leu Gln Ala Leu His Phe Asn Ala
Ile Arg 130 135 140 Gly Gln Asp Gly Arg Met Val Asn Met Ser Val Pro
Ile Val Leu Ser 145 150 155 160 Val Gly Asp Ala Gln Arg Arg Ala Ile
Gln Ala Asp Gly Ala Thr Arg 165 170 175 Val Ala Leu Val Asp Glu Arg
Asp Arg Pro Ile Ala Val Leu Ser Asp 180 185 190 Ile Glu Ile Tyr Lys
His Asn Lys Glu Glu Arg Val Ala Arg Thr Trp 195 200 205 Gly Thr Thr
Ala Pro Gly Leu Pro Tyr Val Glu Glu Ala Ile Thr Asn 210 215 220 Ala
Gly Asp Trp Leu Val Gly Gly Asp Leu Glu Val Ile Glu Pro Ile 225 230
235 240 Lys Tyr Asn Asp Gly Leu Asp Gln Tyr Arg Leu Ser Pro Ala Gln
Leu 245 250 255 Arg Glu Glu Phe Ala Arg Arg Asn Ala Asp Ala Val Phe
Ala Phe Gln 260 265 270 Leu Arg Asn Pro Val His Asn Gly His Ala Leu
Leu Met Thr Asp Thr 275 280 285 Arg Lys Arg Leu Leu Glu Met Gly Tyr
Lys Asn Pro Val Leu Leu Leu 290 295 300 His Pro Leu Gly Gly Phe Thr
Lys Ala Asp Asp Val Pro Leu Ser Trp 305 310 315 320 Arg Met Lys Gln
His Glu Lys Val Leu Glu Glu Gly Val Leu Asn Pro 325 330 335 Glu Ser
Thr Val Val Ala Ile Phe Pro Ser Pro Met His Tyr Ala Gly 340 345 350
Pro Thr Glu Val Gln Trp His Ala Lys Ala Arg Ile Asn Ala Gly Ala 355
360 365 Asn Phe Tyr Ile Val Gly Arg Asp Pro Ala Gly Met Ser His Pro
Thr 370 375 380 Glu Lys Arg Asp Leu Tyr Asp Ala Asp His Gly Lys Lys
Val Leu Ser 385 390 395 400 Met Ala Pro Gly Leu Glu Arg Leu Asn Ile
Leu Pro Phe Lys Val Ala 405 410 415 Ala Tyr Asp Thr Lys Gln Lys Lys
Met Asp Phe Phe Asp Pro Ser Arg 420 425 430 Lys Asp Asp Phe Leu Phe
Ile Ser Gly Thr Lys Met Arg Thr Leu Ala 435 440 445 Lys Asn Arg Glu
Ser Pro Pro Asp Gly Phe Met Cys Pro Gly Gly Trp 450 455 460 Lys Val
Leu Val Glu Tyr Tyr Asp Ser Leu Val Pro Ser Glu Gly Ser 465 470 475
480 Ser Lys Leu Arg Glu Pro Val Ala Ala 485
* * * * *